It has long been known that enzymes are catalysts par excellence. Not only do enzymes vastly increase the reaction rate over that of the uncatalyzed counterpart, but enzymes generally show a very high selectivity both in the transformation catalyzed and in the substrate acted upon. Thus, a relatively small structural change may convert a compound from an enzyme substrate, i.e., a material in which a particular transformation is catalyzed by an enzyme, to a nonsubstrate, i.e., material in which the same transformation is not substantially affected by the same enzyme. The high selectivity manifested by enzymes is not limited only to gross structural changes in the substrate but also to much more subtle differences such as the "handedness," or chirality, of the substrate or product being formed. Being chiral molecules themselves, enzymes generally readily distinguish between enantiomeric substrates, often showing vast differential rate constants for the reaction catalyzed and/or in the formation of enantiomeric products. Additionally, enzymes as catalysts often are remarkably selective with respect to reaction conditions, such as pH, and are effective at or near room temperature. Given these attributes, it is understandable that the preparation of synthetic enzymes has been a continuing goal of chemists and biochemists.
However desirable may be the preparation of synthetic enzymes its achievement has remained largely a dream rather than a reality. This situation undoubtedly arises from the sheer magnitude of the problem. But as the mechanisms of enzymatic reactions have been clarified, as the structures of enzymes have been elucidated, and as the relation between structure, rate, and selectivity has yielded to understanding, the nebulous outlines of solutions to the general problem of synthetic enzyme preparation have been increasingly better defined. Efforts to prepare synthetic enzymes are based on the recognition that enzymatic activity is associated with a three-dimensional structure providing a cavity which binds a substrate in proximity to a moiety which interacts strongly with a particular portion of the substrate. Thus, prior efforts may be classified broadly as being directed either toward providing a cavity which strongly and/or selectively binds substrates, or placing a reactive moiety in the necessary spatial proximity to the substrate site being transformed. Exemplifying the former approach is the use of cyclodextrins as hydrophobic cavities which extract from aqueous solutions those organic molecules having the correct shape. See, e.g., R. Breslow, Science, 218, 532 (1982). Exemplifying the second approach is the "remote functionalization" approach where a template in a molecule controls the site of reaction. Idem., ibid.
Most efforts directed toward constructing enzyme mimics have concentrated on hydrolases, and more particularly esterases. Hydrolases may be broadly defined as molecules which catalyze the hydrolysis of such functional groups as esters, amides, imides, imines, and so forth, whereas esterases are a subgroup which catalyze the hydrolysis of esters. Molecules with a good binding cavity based on macrocyclic structures and providing high rate accelerations have been studied by Cram, J. Am. Chem. Soc., 105, 135 (1983) and by Breslow, J. Am. Chem. Soc., 103, 154 (1981) as esterases. However, the mechanism of ester hydrolysis involves transfer of the acyl group from the ester to the esterase, and in the aforementioned studies the acyl group remains covalently bound to the esterase precluding further catalysis. Stated differently, the esterases of Cram and Breslow remain permanently acylated and no turnover is possible. Consequently, such esterases are in a real sense not catalysts, since they are permanently transformed during the reaction. Polyethylenimines with attached imidazole groups have been shown by Klotz, Proc. Natl. Acad. Sci., 68 (2), 263 (1971) to catalyze ester hydrolysis with turnover for some substrates but the rate enhancement and binding capability are only moderate.
The reaction mechanism of esterases, as representative of hydrolases, can be generally depicted as follows. ##STR1## The first stage is a very fast equilibrium binding of the substrate X-Y to the enzyme EH, generally through a hydrophobic cavity, characterized by an equilibrium constant 1/K.sub.s =k.sub.1 /k.sub.-1, usually called the binding constant. Its reciprocal, K.sub.s, is the dissociation constant of the bound substrate. The second stage is the transfer of an acyl group of the substrate ester to the enzyme with concommitant production of an alcohol, XH, in a reaction whose rate constant is k.sub.2. The third and last stage is the hydrolysis of the acylated enzyme characterized by the rate constant k.sub.3. Usually, k.sub.1,k.sub.-1 &gt;&gt;k.sub.2,k.sub.3. Turnover, i.e., regeneration of free enzyme, is determined by the slower of k.sub.2,k.sub.3. If k.sub.3 is much less than k.sub.2 then k.sub.3 is the rate of turnover, the compound EH is not regenerated in the time frame of substrate hydrolysis, and "enzyme" concentration changes during hydrolysis, which is a departure from the conventional concept of a "catalyst." Conversely, if k.sub.3 is much greater than k.sub.2 (high turnover) the compound EH is regenerated far more rapidly than it is acylated, and the rate of turnover is determined by k.sub.2. Finally, the quantity k.sub.2 /K.sub.M, where K.sub.M =(k.sub.-1 +k.sub.2)/k.sub.1 =K.sub.s +k.sub.2 /k.sub.1, is usually referred to as the catalytic constant.
Whereas the prior efforts referred to above have afforded systems either with (1) low turnover or (2) low binding ability and rate enhancement, the claims herein are based on the discovery that a very large class of molecules, many readily derivable from naturally occurring proteins, have a cavity manifesting good binding propensity with organic substrates and which further have active residues which promote or assist hydrolysis, especially of esters. Such molecules exhibit high turnover with many substrates, and because there is such a large class of such enzyme-like molecules (referred to herein as semisynthetic enzymes), there is a good probability of finding a semisynthetic enzyme-substrate pair in which the system is truly catalytic. One large class of semisynthetic enzymes of this invention are heme proteins from which the heme portion has been removed.